Establishment Of A Technique For Rapid Detection Of Forest Encephalitis Virus

Objective Forest Encephalitis virus((Forest encephalitis virus,FEV),also known as tick-borne encephalitis virus(tick-borne encephalitis virus,TBEV),is a virulent virus of the flavivirus genus of Flaviridae.Its genome is a single-stranded positive-stranded RNA of about 11 kb.Transmission by tick bites can cause a natural epidemic disease in forest areas,namely forest encephalitis(Forest encephalitis,FE).The main clinical symptoms of infected people are characterized by lesions of the central nervous system,which can even lead to death and seriously endanger people’s health and safety.In recent years,the frequent forest garrison training of the army and the increase in the logging activities of forestry personnel,coupled with the rise of forest tours and field trips,have greatly increased the probability of being bitten by ticks,thus increasing the risk of FEV infection.In order to meet the field troops,community grass-roots,hospital bedside and other medical resources under the lack of point-of-care testing of forest encephalitis virus,to achieve early detection of FE epidemic situation,early prevention and control,early treatment,this study established reverse transcriptase polymerase spiral reaction(RT-PSR).Methods The FEV NS1 gene fragment was artificially synthesized,ligated with PGM-T vector,introduced into DH5 α competent cells for cloning,and the plasmid was extracted by shaking bacteria.The extracted plasmid was linearized by Sal I single enzyme digestion,gel cutting,recovery and purification.The purified linearized plasmid was transcribed into RNA fragment by in vitro transcription,which was added to RNA preservation solution at-70 ℃.Then two specific spiral primers Ft/Bt,were designed for the FEV NS1 gene fragment,that is,the plant sequence(Nr/N)was added to the 5 ’end of the forward and backward primers of PCR,and the Nr and N sequences were opposite to each other.Reverse transcriptase polymerase spiral reaction(RT-PSR)was carried out at 65 ℃ to identify whether the reaction product was FEV NS1.Then the reaction conditions were optimized by single factor variation method,including temperature,BST 3.0DNA polymerase concentration,betaine concentration,Mg SO4 concentration,d NTPs concentration and reaction time.After determining the optimum reaction conditions of RT-PSR,the FEV RNA standard samples were diluted 10 times gradient,which were 10-1,10-2,10-3,10-4,10-5,10-6,10-7,10-8,respectively.The sensitivity of RT-PSR and PCR reaction was compared.Using previously synthesized fusion gene(West Nile virus,Japanese Encephalitis virus,Murray Valley Encephalitis virus),DNase/RNase-Free Water,FEV RNA standard samples were detected by RT-PSR under the same reaction conditions to verify the specificity of this method.Whole blood simulated samples were used to verify the ability of this method to detect infected whole blood.Result The electrophoresis results of the 25μL reaction system established in this experiment showed that there were obvious ladder bands,and the sequencing results were consistent with the FEV NS1 sequence published by Genebank.The optimum reaction conditions for FEV detection by RT-PSR were as follows: 6U BST 3.0 DNA polymerase,0.4M betaine,8m M Mg SO4,1.8m M d NTPs,1μL RNA template,Ft/Bt1.6μM,IF/IB 0.8μM,aseptic aseptic enzyme water 25μL,reaction in 61 ℃ constant temperature metal bath for 35 min,after the end of the reaction,1μL of 1:20 diluted SYBR Green I was added.The results can be directly interpreted by the color changes of SYBR Green I before and after the reaction.Only the electrophoresis results containing FEV RNA standard showed obvious trapezoidal bands and SYBR Green I could observe the color change with high specificity.The detection limit of this method is 107.341 × 10-6ng/ μ L,and the detection limit of PCR method is 107.341 ×10-4ng/μL.the detection limit of RT-PSR is 100 times that of PCR.The coincidence rate of whole blood simulation sample was 100%,and the sensitivity was 107.341 ×10-5ng/ μ L.Conclusion The RT-PSR method established in this experiment does not need to reverse RNA in advance,but only needs Bst 3.0DNA polymerase to realize reverse transcription and nucleic acid amplification at the same time at constant temperature.The rapid amplification of FEV is realized by specific spiral primers,and the detection results only need to be determined by adding SYBR Green I indicator and observing the color changes before and after the indicator reaction with naked eyes to determine whether it is a negative reaction or a positive reaction.It only takes 35 minutes,and it has the characteristics of simple,fast,high specificity and high sensitivity.This method can be used to detect trace FEV in whole blood,and can really realize the rapid detection of FEV in field troops and other grass-roots sites.